Heart failure is the leading cause of death in the western world and is known to affect over 550,000 individuals every year. Heart failure is often preceded by hypertrophy, in which the heart wall thickens via growth of the individual myocytes. Our long-term objective is to understand the underlying molecular changes in protein expression, and the temporal resolution of this process, as this knowledge holds the key to ultimately effectively treating and preventing heart failure. These functional and molecular changes currently remain incompletely understood and are only sparsely documented in animal models larger than mice and rats. Recent data led us to hypothesize that the mechanism of developing hypertrophy prior to end-stage failure is critically dependent on the bilateral feedback between the function and expression of myofilament proteins and proteins involved in calcium handling. To understand the progression of the disease, it is thus critically important to identify the earliest changes that appear. In order to assess these temporal changes that occur during the heart's progression from normal to the hypertrophied stage we have developed a novel in vitro system. In this system we use a multi-day culture of functionally contracting multicellular preparations from the rabbit, which enable us to analyze the protein expression and contractile function and regulation throughout the progression from healthy myocardium to diseased myocardium. In aim 1 we will determine the changes in contractile function and regulation during the development of compensatory hypertrophy. In aim 2 we will elucidate the protein expression profile of these multicellular preparations during the progression from normal function to compensatory hypertrophy through the analysis of protein expression and protein phosphorylation. When combined, the temporal resolution of changes in function and protein expression and phosphorylation will give us critical insight into the mechanism of the development of hypertrophy in tissue of a large mammal under near physiological conditions. PUBLIC HEALTH RELEVANCE: The studies proposed will allow for the collection of data to enable the resolution, in a temporal manner, the changes that occur in protein expression and contractile function, as well as their interaction, during the development of hypertrophy. The outcome of the studies will provide crucial insight into the progression of this disease increasing the probability of early detection, and allow us to strategize hypothesis-driven treatment strategies for patients suffering from heart failure.